10 research outputs found
Homologous PNA Hybridization to Noncanonical DNA G‑Quadruplexes
Potential
guanine (G) quadruplex-forming sequences (QFSs) found
throughout the genomes and transcriptomes of organisms have emerged
as biologically relevant structures. These G-quadruplexes represent
novel opportunities for gene regulation at the DNA and RNA levels.
Recently, the definition of functional QFSs has been expanding to
include a variety of unconventional motifs, including relatively long
loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts.
We have identified a QFS within the 25S rDNA gene from <i>Saccharomyces
cerevisae</i> that features a long loop separating the two 3′-most
G-tracts. An oligonucleotide based on this sequence, QFS3, folds into
a stable G-quadruplex in vitro. We have studied the interaction between
QFS3 and several loop mutants with a small, homologous (G-rich) peptide
nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex.
The PNA successfully invades the DNA quadruplex target to form a stable
heteroquadruplex, but with surprisingly high PNA:DNA ratios based
on surface plasmon resonance and mass spectrometric results. A model
for high stoichiometry PNA–DNA heteroquadruplexes is proposed,
and the implications for quadruplex targeting by G-rich PNA are discussed
Hybridization of G‑Quadruplex-Forming Peptide Nucleic Acids to Guanine-Rich DNA Templates Inhibits DNA Polymerase η Extension
The guanine quadruplex (G-quadruplex)
is a highly stable secondary
structure that forms in G-rich repeats of DNA, which can interfere
with DNA processes, including DNA replication and transcription. We
showed previously that short guanine-rich peptide nucleic acids (PNAs)
can form highly stable hybrid quadruplexes with DNA. We hypothesized
that such structures would provide a stronger block to polymerase
extension on G-rich templates than a native DNA homoquadruplex because
of the greater thermodynamic stability of the PNA–DNA hybrid
structures. To test this, we analyzed the DNA primer extension activity
of polymerase η, a translesion polymerase implicated in synthesis
past G-quadruplex blocks, on DNA templates containing guanine repeats.
We observed a PNA concentration-dependent decrease in the level of
polymerase η extension to the end of the template and an increase
in the level of polymerase η inhibition at the sequence prior
to the G-rich repeats. In contrast, the addition of a complementary
C-rich PNA that hybridizes to the G-rich repeats by Watson–Crick
base pairing led to a decrease in the level of polymerase inhibition
and an increase in the level of full-length extension products. The
G-quadruplex-forming PNA exhibited inhibition (IC<sub>50</sub> = 16.2
± 3.3 nM) of polymerase η DNA synthesis on the G-rich templates
stronger than that of the established G-quadruplex-stabilizing ligand
BRACO-19 (IC<sub>50</sub> = 42.5 ± 4.8 nM). Our results indicate
that homologous PNA targeting of G-rich sequences creates stable PNA–DNA
heteroquadruplexes that inhibit polymerase η extension more
effectively than a DNA homoquadruplex. The implications of these results
for the potential development of homologous PNAs as therapeutics for
halting proliferating cancer cells are discussed
Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule
The cyanine dye thiazole orange (TO) is a well-known fluorogenic
stain for DNA and RNA, but this property precludes its use as an intracellular
fluorescent probe for non-nucleic acid biomolecules. Further, as is
the case with many cyanines, the dye suffers from low photostability.
Here, we report the synthesis of a bridge-substituted version of TO
named α-CN-TO, where the central methine hydrogen of TO is replaced
by an electron withdrawing cyano group, which was expected to decrease
the susceptibility of the dye toward singlet oxygen-mediated degradation.
An X-ray crystal structure shows that α-CN-TO is twisted drastically
out of plane, in contrast to TO, which crystallizes in the planar
conformation. α-CN-TO retains the fluorogenic behavior of the
parent dye TO in viscous glycerol/water solvent, but direct irradiation
and indirect bleaching studies showed that α-CN-TO is essentially
inert to visible light and singlet oxygen. In addition, the twisted
conformation of α-CN-TO mitigates nonspecific binding and fluorescence
activation by DNA and a previously selected TO-binding protein and
exhibits low background fluorescence in HeLa cell culture. α-CN-TO
was then used to select a new protein that binds and activates fluorescence
from the dye. The new α-CN-TO/protein fluoromodule exhibits
superior photostability to an analogous TO/protein fluoromodule. These
properties indicate that α-CN-TO will be a useful fluorogenic
dye in combination with specific RNA and protein binding partners
for both in vitro and cell-based applications. More broadly, structural
features that promote nonplanar conformations can provide an effective
method for reducing nonspecific binding of cationic dyes to nucleic
acids and other biomolecules
Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule
The cyanine dye thiazole orange (TO) is a well-known fluorogenic
stain for DNA and RNA, but this property precludes its use as an intracellular
fluorescent probe for non-nucleic acid biomolecules. Further, as is
the case with many cyanines, the dye suffers from low photostability.
Here, we report the synthesis of a bridge-substituted version of TO
named α-CN-TO, where the central methine hydrogen of TO is replaced
by an electron withdrawing cyano group, which was expected to decrease
the susceptibility of the dye toward singlet oxygen-mediated degradation.
An X-ray crystal structure shows that α-CN-TO is twisted drastically
out of plane, in contrast to TO, which crystallizes in the planar
conformation. α-CN-TO retains the fluorogenic behavior of the
parent dye TO in viscous glycerol/water solvent, but direct irradiation
and indirect bleaching studies showed that α-CN-TO is essentially
inert to visible light and singlet oxygen. In addition, the twisted
conformation of α-CN-TO mitigates nonspecific binding and fluorescence
activation by DNA and a previously selected TO-binding protein and
exhibits low background fluorescence in HeLa cell culture. α-CN-TO
was then used to select a new protein that binds and activates fluorescence
from the dye. The new α-CN-TO/protein fluoromodule exhibits
superior photostability to an analogous TO/protein fluoromodule. These
properties indicate that α-CN-TO will be a useful fluorogenic
dye in combination with specific RNA and protein binding partners
for both in vitro and cell-based applications. More broadly, structural
features that promote nonplanar conformations can provide an effective
method for reducing nonspecific binding of cationic dyes to nucleic
acids and other biomolecules
In Vitro Reversible Translation Control Using γPNA Probes
On-demand
regulation of gene expression in living cells is a central
goal of chemical biology and antisense therapeutic development. While
significant advances have allowed regulatory modulation through inserted
genetic elements, on-demand control of the expression/translation
state of a given native gene by complementary sequence interactions
remains a technical challenge. Toward this objective, we demonstrate
the reversible suppression of a luciferase gene in cell-free translation
using Watson–Crick base pairing between the mRNA and a complementary
γ-modified peptide nucleic acid (γPNA) sequence with a
noncomplementary toehold. Exploiting the favorable thermodynamics
of γPNA−γPNA interactions, the antisense sequence
can be removed by hybridization of a second, fully complementary γPNA,
through a strand displacement reaction, allowing translation to proceed.
Complementary RNA is also shown to displace the bound antisense γPNA,
opening up possibilities of in vivo regulation by native gene expression
RNA G‑Quadruplex Invasion and Translation Inhibition by Antisense γ‑Peptide Nucleic Acid Oligomers
We have examined the abilities of
three complementary γ-peptide
nucleic acid (γPNA) oligomers to invade an RNA G-quadruplex
and potently inhibit translation of a luciferase reporter transcript
containing the quadruplex-forming sequence (QFS) within its 5′-untranslated
region. All three γPNA oligomers bind with low nanomolar affinities
to an RNA oligonucleotide containing the QFS. However, while all probes
inhibit translation with low to midnanomolar IC<sub>50</sub> values,
the γPNA designed to hybridize to the first two G-tracts of
the QFS and adjacent 5′-overhanging nucleotides was 5–6
times more potent than probes directed to either the 3′-end
or internal regions of the target at 37 °C. This position-dependent
effect was eliminated after the probes and target were preincubated
at an elevated temperature prior to translation, demonstrating that
kinetic effects exert significant control over quadruplex invasion
and translation inhibition. We also found that antisense γPNAs
exhibited similarly potent effects against luciferase reporter transcripts
bearing QFS motifs having G<sub>2</sub>, G<sub>3</sub>, or G<sub>4</sub> tracts. Finally, our results indicate that γPNA oligomers
exhibit selectivity and/or potency higher than those of other antisense
molecules such as standard PNA and 2′-OMe RNA previously reported
to target G-quadruplexes in RNA
Fluoromodules Consisting of a Promiscuous RNA Aptamer and Red or Blue Fluorogenic Cyanine Dyes: Selection, Characterization, and Bioimaging
An RNA aptamer selected for binding
to the fluorogenic cyanine
dye, dimethylindole red (DIR), also binds and activates another cyanine,
oxazole thiazole blue (OTB), giving two well-resolved emission colors.
The aptamer binds to each dye with submicromolar <i>K</i><sub>D</sub> values, and the resulting fluoromodules exhibit fluorescence
quantum yields ranging from 0.17 to 0.51 and excellent photostability.
The aptamer was fused to a second aptamer previously selected for
binding to the epidermal growth factor receptor (EGFR) to create a
bifunctional aptamer that labels cell-surface EGFR on mammalian cells.
The fluorescent color of the aptamer-labeled EGFR can be switched
between blue and red in situ simply by exchanging the dye in the medium.
The promiscuity of the aptamer can also be used to distinguish between
cell-surface and internalized EGFR on the basis of the addition of
red or blue fluorogen at different times
Bright Fluorescent Nanotags from Bottlebrush Polymers with DNA-Tipped Bristles
Bright
signal outputs are needed for fluorescence detection of
biomolecules at their native expression levels. Increasing the number
of labels on a probe often results in crowding-induced self-quenching
of chromophores, and maintaining the function of the targeting moiety
(e.g., an antibody) is a concern. Here we demonstrate a simple method
to accommodate thousands of fluorescent dye molecules on a single
antibody probe while avoiding the negative effects of self-quenching.
We use a bottlebrush polymer from which extend hundreds of duplex
DNA strands that can accommodate hundreds of covalently attached and/or
thousands of noncovalently intercalated fluorescent dyes. This polymer–DNA
assembly sequesters the intercalated fluorophores against dissociation
and can be tethered through DNA hybridization to an IgG antibody.
The resulting fluorescent nanotag can detect protein targets in flow
cytometry, confocal fluorescence microscopy, and dot blots with an
exceptionally bright signal that compares favorably to commercially
available antibodies labeled with organic dyes or quantum dots
A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red
Novel fluorescent tools such as green fluorescent protein
analogues and fluorogen activating proteins (FAPs) are useful in biological
imaging for tracking protein dynamics in real time with a low fluorescence
background. FAPs are single-chain variable fragments (scFvs) selected
from a yeast surface display library that produce fluorescence upon
binding a specific dye or fluorogen that is normally not fluorescent
when present in solution. FAPs generally consist of human immunoglobulin
variable heavy (V<sub>H</sub>) and variable light (V<sub>L</sub>)
domains covalently attached via a glycine- and serine-rich linker.
Previously, we determined that the yeast surface clone, V<sub>H</sub>-V<sub>L</sub> M8, could bind and activate the fluorogen dimethylindole
red (DIR) but that the fluorogen activation properties were localized
to the M8V<sub>L</sub> domain. We report here that both nuclear magnetic
resonance and X-ray diffraction methods indicate the M8V<sub>L</sub> forms noncovalent, antiparallel homodimers that are the fluorogen
activating species. The M8V<sub>L</sub> homodimers activate DIR by
restriction of internal rotation of the bound dye. These structural
results, together with directed evolution experiments with both V<sub>H</sub>-V<sub>L</sub> M8 and M8V<sub>L</sub>, led us to rationally
design tandem, covalent homodimers of M8V<sub>L</sub> domains joined
by a flexible linker that have a high affinity for DIR and good quantum
yields
Antitumor Effects of EGFR Antisense Guanidine-Based Peptide Nucleic Acids in Cancer Models
Peptide nucleic acids have emerged over the past two
decades as a promising class of nucleic acid mimics because of their
strong binding affinity and sequence selectivity toward DNA and RNA,
and resistance to enzymatic degradation by proteases and nucleases.
While they have been shown to be effective in regulation of gene expression <i>in vitro</i>, and to a small extent <i>in vivo</i>, their full potential for molecular therapy has not yet been fully
realized due to poor cellular uptake. Herein, we report the development
of cell-permeable, guanidine-based peptide nucleic acids targeting
the epidermal growth factor receptor (EGFR) in preclinical models
as therapeutic modality for head and neck squamous cell carcinoma
(HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16
nucleotides in length, designed to bind to EGFR gene transcript elicited
potent antisense effects in HNSCC and NSCLC cells in preclinical models.
When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up
by several tissues including the xenograft tumor. Systemic administration
of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with
similar efficacies as the FDA-approved EGFR inhibitors: cetuximab
and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA
is effective against the constitutively active EGFR vIII mutant implicated
in cetuximab resistance. Our data reveals that GPNA is just as effective
as a molecular platform for treating cetuximab resistant cells, demonstrating
its utility in the treatment of cancer